Binding agents to pre-fusion state sars-cov-2 spike protein

ABSTRACT

Disclosed is producing recombinant SARS-CoV-2 spike protein in a pre-fusion state, using furin knock out or knockdown mammalian cells (such as HEK293, CHO or other mammalian cells) and using them to generate antibodies and related binding agents. The antibodies/binding agents can be used in SARS-CoV-2 detection assays or in diagnosis of active or prior infection with SARS-CoV-2; in prophylaxis or as a therapeutic; or for prophylactic or therapeutic use against coronaviruses related to SARS-CoV-2.

SEQUENCE LISTING STATEMENT

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety.

BACKGROUND

For the coronavirus SARS-CoV-2, which is responsible for the COVID-19pandemic, only limited treatment options having limited efficacy areavailable. It is known to primarily enter human cells by binding of itsspike protein to the receptor angiotensin converting enzyme 2 (ACE2).

SARS-CoV-2 has four structural proteins, known as the S (spike), E(envelope), M (membrane), and N (nucleocapsid) proteins. The N proteinholds the RNA genome, and the S, E, and M proteins together create theviral envelope. The spike (“5”) protein is responsible for allowing thevirus to attach to and fuse with the membrane of a host cell.

The S protein is a class I fusion protein; which are known to exist astrimers in their pre-fusion and post-fusion states. The S protein 51subunit mediates cellular attachment, and the S2 subunit is involved infusion which allows viral genome entry into the cell. The S protein hastwo states, a pre-fusion state and a mature/active form, achieved afterproteolytic cleavage and activation.

Recombinant SARS-CoV-2 structural proteins are essential for antibody,vaccine and drug development. Recombinant wild-type S-ECD (extracellulardomain) is challenging to produce and very unstable. Most of therecombinant S-ECDs on the market contain a mutation to avoid proteasecleavage, making the recombinant version different from wild-type andpotentially less useful in research or therapy.

Proteolytic cleavage of the S protein can occur in the constitutivesecretory pathway of infected cells or during viral entry into targetcells, and is essential for viral infectivity.

Furin is a processing enzyme that cleaves substrate proteins into theirmature/active forms. Substrates of furin include blood clotting factors,serum proteins and growth factor receptors as well as the viral spikeproteins. Furin belongs to the subtilisin-like proprotein convertasefamily. The members of this family are proprotein convertases thatprocess latent precursor proteins into their biologically activeproducts. Furin is enriched in the Golgi apparatus, where it functionsto cleave other proteins into their mature/active forms. Furin isbelieved to be one of the proprotein convertases for the S protein.

Therefore, inhibition or disruption of cleavage of the S protein byfurin may allow production of the S protein in a pre-fusion state(referred to as S-ECD-PFS). S-ECD-PFS can be used as an antigen togenerate antibodies for use in detection assays or in diagnosis, or togenerate antibodies and related binding agents to SARS-CoV-2 for use inpassive immunization or therapy.

SUMMARY

The invention relates to using furin knock out or knockdown mammaliancells (such as HEK293, CHO or other mammalian cells) which producerecombinant SARS-CoV-2 spike protein in a pre-fusion state, to generateantibodies and related binding agents. An exemplary method of generatingS-ECD-PFS and confirming its activity is summarized as follows:

-   -   1. Use a CRISPR Cas9 protocol to knock out the furin gene in        mammalian cells, such as HEK293 cells;    -   2. Transfect expression vectors for the S-ECD gene into the        furin−/− cells;    -   3. Purify recombinant S-ECD-PFS, preferably using a nickel        column; and    -   4. Measure S-ECD-PFS' bio-activity, for example, by determining        its binding ability to recombinant Human ACE2 in a functional        ELISA.

The recombinant S-ECD-PFS can be used as an antigen to generateantibodies/binding molecules for use in detection assays (e.g., in bloodor tissues for transfusion or transplantation) or in diagnosis of activeor prior infection with SARS-CoV-2. The recombinant S-ECD-PFS can alsobe used as an antigen to generate antibodies/binding molecules toSARS-CoV-2 for use in passive immunization or other therapy, againstSARS-CoV-2 or the following viruses related to SARS-CoV-2: CoV-ZXC21(MG772934), SARSCoV (NC_004718.3), SARS-like BM4821(MG772934), HCoV-OC43(AY391777), HKU9-1 (EF065513), HCoV-NL63 (KF530114.1), HCoV229E(KF514433.1), MERS-CoV (NC019843.3), and HKU1 (NC_006577.2).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the domain organization of the SARS-CoV-2 spike proteinand illustrates the furin cleavage site.

FIG. 2 illustrates the bands in the gel from proteolytic processing andseparation of recombinant S-ECD-PFS when expressed in HEK293.

FIG. 3a illustrates the differences between the furin gene wild type(“WT”) and the knock out gene expressed in HEK293 clones 30-18 and30-28. The sequences shown are WT, wild type (SEQ ID NO: 1) and KO,knock out (SEQ ID NO: 2), with the WT insert separately shown (SEQ IDNO: 3).

FIG. 3b is a Western blot, showing that furin was not present in themedia containing HEK293 clones 30-18 and 30-28 (right-hand column) basedon absence of agglutination in the right-hand column following adding aanti-furin antibody to the media, with the left-hand column includingcells expressing WT furin, and the second right-hand includinganti-actin, as controls.

FIG. 3c is a gel showing S-ECD-PFS as a single major band (col. 2) whenexpressed in FURIN gene knockout HEK293 cells, with HEK293 cellsexpressing furin WT (col. 1) as control.

FIG. 4 is a gel showing media fractions (cols. 1-5) from the furin−/−cells, following protein purification with a nickel column

FIG. 5 shows results from an ACE2 binding ELISA for the recombinantS-ECD-PFS, and for a conventional, currently marketed recombinant S-ECD.

DETAILED DESCRIPTION

The singular forms “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise.

A “binding agent” refers to all antibodies, antibody fragments orderivatives of antibodies as described below, as well as proteins andmolecules other than proteins, antibodies and fragments or derivativesof antibodies which target S-ECD-PFS, any of which could be modified andtested to become binding agents with high affinity for S-ECD-PFS, usingtechniques similar to those described below.

A “conjugate” includes conjugates with antibodies or binding agents orconjugates with S-ECD-PFS, as determined from the context. A conjugatecan include fusion proteins and proteins or antibodies conjugated withone or more than one polypeptide or antibody or Binding Agent, nucleicacid or chemical compound or a drug carrier. Alternatively, or inaddition, a conjugate can comprise one or more other pharmaceuticallyactive agents or drugs. Examples of such other pharmaceutically activeagents or drugs that may be suitable for use in a conjugate (or includedin a drug carrier) include antibiotics, known antiviral drugs, cytotoxicdrugs, and other antibiotics and drugs known or suspected to inhibit orameliorate Covid 19 infection, including hydroxychloroquine,zithromycin, Remdesivir™, nitric oxide (encapsulated), Ifenprodil,ribavirin, interferon β-1a, interferon-α, other interferons, RecombinantMycobacterium bovis, colchicine, favipiravir, lopinavir, ritonavir,Peginterferon Lambda-1A, and Fenretinide, as well as other antibioticse.g., doxorubicin, vincristine, cisplatin, daunomycin, methotrexate andother anticancer agents such as toxins (such as diphtheria or ricin),cyclophosphamide and other medications, also can be used. alkylatingagents (e.g., cyclophosphamide, melphalan etc.), cytotoxic antibiotics(doxorubicin, epirubicin, bleomycin, mitomycin, methotrexate,capecitabine, gemcitabine, fluorouracil, vinca alkaloids and etoposide(vinblastine, vincristine etc.), platinum compounds (carboplatin,cisplatin, oxaliplatin), taxanes (paclitaxel, docetaxel, etc.),topoisomerase I inhibitors (irinotecan, topotecan, etc.) as well asagents like cyclophosphamide, and combinations thereof. In oneembodiment, the composition can comprise one or more polypeptide(antibody), fusion protein, conjugate, nucleic acid, vector, or cell ofthe invention and one or more other pharmaceutically active agents ordrugs.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine).

A “drug carrier” includes liposomes, polymeric micelles, microspheresand nanoparticles.

The term “linker” refers to a compound or moiety that acts as amolecular bridge to operably link two different molecules (e.g., whereinone portion of the linker binds to a binding agent, and wherein anotherportion of the linker binds to the other molecule(s) in the conjugate).Linkers include, but are not limited to, chemical chains, chemicalcompounds (e.g., reagents), amino acids, and the like. The linkers mayinclude, but are not limited to, homobifunctional linkers,hetero-bifunctional linkers, biostable linkers, and biodegradablelinkers. The linker may be non-planar (e.g., so that the boundcomponents in the conjugate are not rigidly fixed). Heterobifunctionallinkers, contain one end having a first reactive functionality tospecifically link a first molecule, and an opposite end having a secondreactive functionality to specifically link to a second molecule.Depending on such factors as the molecules to be linked, and theconditions in which the linking is performed, the linker may vary inlength and composition for optimizing such properties as preservation ofbiological function stability, resistance to certain chemical and/ortemperature parameters, and of sufficient stereo-selectivity or size.Preferably the linker is a “synthetic peptidic linker” that isdesignated to be rich in glycine, glutamine, and/or serine residues.These residues are arranged e.g. in small repetitive units of up to fiveamino acids. This small repetitive unit may be repeated for two to fivetimes to form a multimeric unit. At the amino- and/or carboxy-terminalends of the multimeric unit up to six additional arbitrary, naturallyoccurring amino acids may be added.

The term S-ECD-PFS refers to SEQ ID NO: 16 and in the context ofpreparation of antibodies/binding agents for therapy, prophylaxis or indetection assays, is to be read as including other related proteins usedin the same way as such antibodies/binding agents, includingantibodies/binding agents made using the techniques described hereinagainst any of: CoV-ZXC21 (MG772934), SARSCoV (NC_004718.3), SARS-likeBM4821(MG772934), HCoV-OC43 (AY391777), HKU9-1 (EF065513), HCoV-NL63(KF530114.1), HCoV229E (KF514433.1), MERS-CoV (NC019843.3), HKU1(NC_006577.2).

The term “sequence identity” refers to the identical amino acids in twosequences compared; such that they can range from 0 to 100% sequenceidentity. In some embodiments, the sequence identity of a relatedprotein can be about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% sequence identity with S-ECD-PFS or theantibodies/binding agents against it.

Binding Agents

The invention includes antibodies (polyclonal and monoclonal) and otherbinding agents targeting S-ECD-PFS, administered for diagnosis,prophylaxis or therapy of SARS-CoV-2 or related pathogen infection, orfor detection of SARS-CoV-2 or related pathogens in blood units ortissue samples, or in forensic applications, including diseasemonitoring.

Typically, an antibody has a heavy and light chain. Each heavy and lightchain contains a constant region and a variable region (VH and VL,respectively). The regions are also known as “domains.” Light and heavychain variable regions contain a “framework” region interrupted by threehypervariable regions, also called “complementarity-determining regions”or “CDRs.” The sequences of the framework regions of different light orheavy chains are relatively conserved within a species. The frameworkregion of an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs in three dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a VH CDR3 is located in the variable domain of the heavychain of the antibody in which it is found, whereas a VL CDR1 is theCDR1 from the variable domain of the light chain of the antibody inwhich it is found.

In one preferred embodiment, an anti-S-ECD-PFS monoclonal antibody(usually generated in mice or other rodents) or a fragment thereof, is achimeric, humanized, or human monoclonal antibody. Generally, ahumanized antibody has one or more amino acid residues introduced intoit from a source that is non-human. These non-human amino acid residuesare often referred to as import residues, which are typically taken froman import variable domain. Humanization can be essentially performedfollowing the methods described in Jones et al., Nature 321: 522-525(1986); Riechmann et al., Nature 332: 323-327 (1988); or Verhoeyen etal., Science 239: 1534-1536 (1988), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody.Accordingly, such humanized antibodies are chimeric antibodies (U.S.Pat. No. 4,816,567) wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species. In practice, humanized antibodies are typicallyhuman antibodies in which some CDR residues and possibly some FRresidues are substituted by residues from analogous sites in rodentantibodies.

In some embodiments, “human antibody” refers to an immunoglobulincomprising human hypervariable regions in addition to human frameworkand constant regions. Such antibodies can be produced using varioustechniques known in the art. For example in vitro methods involve use ofrecombinant libraries of human antibody fragments displayed onbacteriophage (e.g., McCafferty et al, 1990, Nature 348: 552-554;Hoogenboom & Winter, J. Mol. Biol. 227: 381(1991); and Marks et al, J.Mol. Biol. 222: 581 (1991)), yeast cells (Boder and Wittrup, 1997, NatBiotechnol 15: 553-557), or ribosomes (Hanes and Pluckthun, 1997, ProcNatl Acad Sci USA 94: 4937-4942). Similarly, human antibodies can bemade by introducing human immunoglobulin loci into transgenic animals,e.g., mice in which the endogenous immunoglobulin genes have beenpartially or completely inactivated. Upon challenge, human antibodyproduction is observed, which closely resembles that seen in humans inall respects, including gene rearrangement, assembly, and antibodyrepertoire. This approach is described, e.g., in U.S. Pat. Nos.6,150,584, 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,661,016, and in the following scientific publications: (e.g.,Jakobavits, Drug Deliv Rev. 31: 33-42 (1998), Marks et al,Bio/Technology 10: 779-783 (1992); Lonberg et al, Nature 368: 856-859(1994); Morrison, Nature 368: 812-13 (1994); Fishwild et al, NatureBiotechnology 14: 845-51 (1996); Neuberger, Nature Biotechnology 14: 826(1996); Lonberg &Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

In certain embodiments, the antibody or the fragment thereof disclosedherein comprises or is an F(ab)′2, an Fab, an Fv, or a single-chain Fvfragment of the above anti-S-ECD-PFS antibodies.

In some embodiments, “antibody fragments” means molecules that comprisea portion of an intact antibody, generally the antigen binding orvariable region of the intact antibody. Examples of antibody fragmentsinclude Fab, Fab′, F(ab)′2, and Fv fragments; single domain antibodies(see, e.g., Wesolowski, Med Microbiol Immunol. (2009) 198 (3): 157-74;Saerens, et al., Curr Opin Pharmacol. (2008) 8 (5): 600-8; Harmsen andde Haard, Appl Microbiol Biotechnol. (2007) 77 (1): 13-22));helix-stabilized antibodies (see, e.g., Arndt et al., J Mol Biol 312:221-228 (2001); diabodies (see below); single-chain antibody molecules(“scFvs,” see, e.g., U.S. Pat. No. 5,888,773); disulfide stabilizedantibodies (“dsFvs”, see, e.g., U.S. Pat. Nos. 5,747,654 and 6,558,672),and domain antibodies (“dAbs,” see, e.g., Holt et al., Trends Biotech21(11): 484-490 (2003), Ghahroudi et al., FEBS Lett. 414: 521-526(1997), Lauwereys et al., EMBO J 17: 3512-3520 (1998), Reiter et al., J.Mol. Biol. 290: 685-698 (1999), Davies and Riechmann, Biotechnology, 13:475-479 (2001)).

U.S. Pat. No. 5,932,448 discloses making of bispecific antibodies withFab′ portions joined by a leucine zipper; U.S. Pat. No. 7,538,196,discloses making of bispecific antibodies where portions are joined witha linker; U.S. Pat. No. 8,148,496 discloses a multi-specific Fv antibodyconstruct having at least four variable domains which are linked witheach other via peptide linkers. A bispecific antibody could have one armtargeting ICB and the other arm targeting a tumor or cancer marker.

US Publ'n No. 20170335281 describes making of a genetically modified Tcell expressing a CAR that comprises an antigen binding domain thatbinds to a cancer associated antigen. The same general techniques can beapplied to modify T cells or other immune effector cells to express oneor more of CDR1, CDR2 and CDR3 of an antigen binding domain, for cancertreatment. The antigen binding domain of the CAR polypeptide moleculecan include any antibody, antibody fragment, an scFv, a Fv, a Fab, a(Fab′)₂, a single domain antibody (SDAB, disclosed in WO 9404678 andHamers-Casterman, C. et al. (1993) Nature 363:446-448), a VH or VLdomain, or a VHH domain. Such CAR expressing T cells could be used incombination with the therapy described herein, or the antigen bindingdomain could be an anti-S-ECD-PFS domain.

High Affinity Antibody Variants

Making of Anti-S-ECD-PFS Antibodies with High Affinity

Antibodies can be humanized with retention of high affinity for theantigen and other favorable biological properties. To achieve this goal,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available. Computerprograms are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the recipient andimport sequences so that the desired antibody characteristic, such asincreased affinity for the target antigen(s), is achieved.

Examples of framework region residues to modify include those whichnon-covalently bind target directly (Amit et al. Science 233: 747-753(1986)); interact with/effect the conformation of CDR (Chothia et al. J.Mol. Biol. 196: 901-917 (1987)); and/or participate in the VL-VHinterface (EP 239 400 B1). In certain embodiments, modification of oneor more of such framework region residues results in an enhancement ofthe binding affinity of the antibody for the target of interest.

Nucleic acid molecules encoding amino acid sequence variants areprepared by a variety of methods known in the art. These methodsinclude, but are not limited to, oligonucleotide-mediated (orsite-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis ofan earlier prepared variant or a non-variant version of thespecies-dependent antibody. The preferred method for generating variantsis an oligonucleotide-mediated synthesis. In certain embodiments, theantibody variant will only have a single hypervariable region residuesubstituted, e.g. from about two to about fifteen hypervariable regionsubstitutions.

One method for generating the library of variants is by oligonucleotidemediated synthesis. Three oligonucleotides of approximately 100nucleotides each may be synthesized spanning the entire light chain orheavy chain variable region. Each oligonucleotide may comprise: (1) a 60amino acid stretch generated by the triplet (NNK)20 where N is anynucleotide and K is G or T, and (2) an approximately 15-30 nucleotideoverlap with either the next oligo or with the vector sequence at eachend. Upon annealing of these three oligonucleotides in a PCR reaction,the polymerase will fill in the opposite strand generating a completedouble stranded heavy chain or light chain variable region sequence. Thenumber of triplets may be adjusted to any length of repeats and theirposition within the oligonucleotide may be chosen so as to onlysubstitute amino acids in a given CDR or framework region. By using(NNK), all twenty amino acids are possible at each position in theencoded variants. The overlapping sequence of 5-10 amino acids (15-30nucleotides) will not be substituted, but this may be chosen to fallwithin the stacking regions of the framework, or may substituted by aseparate or subsequent round of synthesis. Methods for synthesizingoligonucleotides are well known in the art and are also commerciallyavailable. Methods for generating the antibody variants from theseoligonucleotides are also well known in the art, e.g., PCR.

The library of heavy and light chain variants, differing at randompositions in their sequence, can be constructed in any expressionvector, such as a bacteriophage, each of which contains DNA encoding aparticular heavy and light chain variant.

Following production of the antibody variants, the biological activityof variant relative to the parent antibody is determined. As notedabove, this involves determining the binding affinity of the variant forthe ICB target. Numerous high-throughput methods exist for rapidlyscreening antibody variants for their ability to bind the target ofinterest.

One or more of the antibody variants selected from this initial screenmay then be screened for enhanced binding affinity relative to theparent antibody. One common method for determining binding affinity isby assessing the association and dissociation rate constants using aBIAcore surface plasmon resonance system (BIAcore, Inc.). A biosensorchip is activated for covalent coupling of the target according to themanufacturer's (BIAcore) instructions. The target is then diluted andinjected over the chip to obtain a signal in response units (RU) ofimmobilized material. Since the signal in RU is proportional to the massof immobilized material, this represents a range of immobilized targetdensities on the matrix. Dissociation data are fit to a one-site modelto obtain koff+/−s.d. (standard deviation of measurements). Pseudo-firstorder rate constant (ks) are calculated for each association curve, andplotted as a function of protein concentration to obtain kon+/−s.e.(standard error of fit). Equilibrium dissociation constants for binding,Kd's, are calculated from SPR measurements as koff/kon. Since theequilibrium dissociation constant, Kd, is inversely proportional tokoff, an estimate of affinity improvement can be made assuming theassociation rate (kon) is a constant for all variants.

The resulting candidate(s) with high affinity may optionally besubjected to one or more further biological activity assays to confirmthat the antibody variant(s) with enhanced binding affinity still retainthe desired therapeutic attributes, as can be tested in the assaysdescribed in the figures above. The optimal antibody variant retains theability to bind the ICB target with a binding affinity significantlyhigher than the parent antibody. The antibody variant(s) so selected maybe subjected to further modifications oftentimes depending upon theintended use of the antibody. Such modifications may involve furtheralteration of the amino acid sequence, fusion to heterologouspolypeptide(s) and/or covalent modifications such as those elaboratedbelow. For example, any cysteine residues not involved in maintainingthe proper conformation of the antibody variant may be substituted,generally with serine, to improve the oxidative stability of themolecule and prevent aberrant cross linking. Conversely, (a) cysteinebond(s) may be added to the antibody to improve its stability(particularly where the antibody is an antibody fragment such as an Fvfragment).

Conjugates of Binding Agents

Conjugates of binding agents with any cytotoxic agents, antibiotics,known antiviral drugs, cytotoxic drugs, and other antibiotics and drugsknown or suspected to inhibit or ameliorate Covid 19 infection notedabove, as well as with drug carriers containing any such cytotoxicagents, antibiotics, etc., can be used in therapy or prophylaxis ofCovid 19.

Such binding agents and methods of conjugating the binding agents to amolecule of interest are well known in the art and have been used in theproduction of antibody conjugates. Some examples of linking groups andconjugation methods are described in US Publication No. 2011/060318(incorporated by reference). The choice of binding agent, coupling(conjugation) technique and linking group for use in the conjugatesdescribed herein is well known.

Binding agents are conjugated via reactive sites on the binding agentsvia a linking group. For example, primary amino groups present on aminoacid residue such as the epsilon amino group of lysine, and the alphaamino group of N-terminal amino acids of proteins can be used asfunctional groups for conjugation. Often it is desirable to convert oneor more primary amino groups of a binding agent to a thiol-containinggroup (e.g., from a cysteine or homocysteine residue), an electrophilicunsaturated group such as a maleimide group, or halogenated group suchas a bromoacetyl group, for conjugation to thiol reactive peptides.Optionally, a primary amino group on the hemagglutinin FIR peptide or ona linker moiety attached to the peptide, can be converted to thethiol-containing group, for coupling with a thiol (sulfhydryl) moiety onthe carrier protein, e.g., by a disulfide bond.

In some embodiments, the conjugation can be achieved, for example, byusing succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate(SMCC), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sSMCC C-C-maleimidocaproyloxyl-sulfosuccinimde ester (sEMCS),bis-diazobenzidine (BDB), N-maleimidobenzoyl-N-hydroxysuccinimide ester(MBS), glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDCI), or N-acetyl homocysteine thiolactone (NAHT).

In the SMCC method, SMCC cross-links the SH-group of a cysteine residueto the amino group of a lysine residue on the binding agent. In the SMCCmethod, the binding agent first is activated by reacting SMCC with aprimary amine (e.g., on a lysine residue of the carrier protein). Theresulting activated binding agent is then separated from any excess SMCCand by-product therefrom, and a cysteine-containing peptide is added.The thiol group of the cysteine adds across the double bond of themaleimide moiety of the SMCC-derivatized binding agent, thus forming acovalent sulfide bond to couple the binding agent to the peptide. If ahemagglutinin FIR peptide does not include a cysteine residue, then acysteine residue should be added to the peptide, preferably at theN-terminus or C-terminus. If the epitope portion of the hemagglutininFIR peptide contains a cysteine or if there is more than one cysteinegroup in the peptide, then another conjugation technique that does notmodify the cysteine residues should be utilized. Since the linkagebetween the binding agent and the peptide should not interfere with theepitope portion of the peptide, the added cysteine residue optionallycan be separated from the hemagglutinin FIR peptide by including one ormore amino acid residues as a spacer. The cysteine, spacer residues, andthe modified SMCC attached to the binding agent constitute the linkinggroup of the hemagglutinin FIR peptide conjugate.

Another simple coupling of a peptide to a binding agent can be achievedwith a carbodiimide crosslinker such as1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC),1-cyclohexyl-2-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate(CMC), and the like to covalently attach carboxyl groups to primaryamine groups. This method is simple and provides a relatively randomorientation that allows for antibody generation against many possibleepitopes. One drawback is that EDC coupling can result in some amount ofpolymerization. This can decrease the solubility of the conjugate, whichcan complicate the handling of the material.

Other coupling agents can be used to conjugate the FIR peptide to thebinding agent, either directly or via a linking group. For example,conjugation can be achieved using isocyanate coupling agents, such as2-morpholinoethylisocyanide; N-acetyl homocysteine thiolactone, whichcan be used to add a thiol group onto a binding agent such as OMPCcoupling with a maleimide or bromoacetyl functionalized peptide; or anyother agents for coupling haptens (potential immunogens) to polypeptidesand proteins, many of which are well known.

Non-specific cross-linking agents and their use are well known in theart. Examples of such reagents and their use include reaction withglutaraldehyde; reaction withN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide, with or withoutadmixture of a succinylated carrier; periodate oxidation of glycosylatedsubstituents followed by coupling to free amino groups of a proteincarrier in the presence of sodium borohydride or sodiumcyanoborohydride; periodate oxidation of non-acylated terminal serineand threonine residues forming terminal aldehydes which can then bereacted with amines or hydrazides creating a Schiff base or a hydrazone,which can be reduced with cyanoborohydride to secondary amines;diazotization of aromatic amino groups followed by coupling on tyrosineside chain residues of the protein; reaction with isocyanates; orreaction of mixed anhydrides. The linkers can be supplemented andextended with spacer groups, such as additional amino acid residues,adipic acid dihydrazide, and the like.

Typical spacer peptide groups for use in conjugation of the FIR peptideto the binding agent include single amino acids (e.g., Cys) and shortpeptide sequences (i.e., short non-hemagglutinin FIR peptide sequences)attached to the FIR peptide, e.g., a lysine containing peptide, acysteine-containing peptide, and the like. Some preferred linking groupscomprise a sulfide bond (e.g., as in SMCC and related coupling methods).Some preferred linking groups include a Cys residue bound to thesuccinimido moiety through the sulfhydryl side chain thereof which isbound the N-terminus of the FIR peptide by a peptide bond. The1-carbonyl group on the cyclohexyl moiety of Formula I is bound to aprimary amine on the binding agent by an amide bond.

In some embodiments, the peptide conjugates include a singlehemagglutinin FIR peptide attached to the binding agent, while in otherembodiments, two or more hemagglutinin FIR peptides can be attached tothe binding agent.

Formulations for. In Vivo Use

I. Dosing of the Binding Agents and Other Active Ingredients

1. Formulations

The dosages of binding agents or conjugates for treating or prophylaxisof SARS-CoV2 can be determined as follows. The conjugates can includepolyethylene glycol, immunoglobulin Fc fragments, or collagen, albuminand other proteins which are linked to a base molecule, and antibodies(including monoclonal antibodies and fragments thereof). Any compositionor compound that can simulate the biological response associated withthe binding of ACE-2 receptors can be used. General details ontechniques for formulation and administration are well-described in thescientific literature (see, e.g., Remington's Pharmaceutical Sciences,Maack Publishing Co., Easton Pa.).

The formulations containing pharmaceutically active products used in themethods of the disclosure can be formulated for administration in anyconventionally acceptable way including, but not limited to,intravenously, subcutaneously, intramuscularly, sublingually, topically,orally and via inhalation. Illustrative examples are set forth below.

When the formulations are delivered by intravenous injection, theformulations containing pharmaceutically active ligands can be in theform of a sterile injectable preparation, such as a sterile injectableaqueous or oleaginous suspension. This suspension can be formulatedaccording to the known art using those suitable dispersing or wettingagents and suspending agents which have been mentioned above. Thesterile injectable preparation can also be a sterile injectable solutionor suspension in a nontoxic parenterally-acceptable diluent or solvent.Among the acceptable vehicles and solvents that can be employed arewater and Ringer's solution, an isotonic sodium chloride. In addition,sterile fixed oils can conventionally be employed as a solvent orsuspending medium. For this purpose any bland fixed oil can be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid can likewise be used in the preparation of injectables.

Pharmaceutical formulations for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical formulations to be formulated in unit dosage forms astablets, pills, powder, capsules, liquids, lozenges, gels, syrups,slurries, suspensions, etc., suitable for ingestion by the patient,which would often include an enteric coating to prevent destruction ofthe ligand in the highly acidic environment of the stomach.Pharmaceutical preparations for oral use can be combinations of ligandswith a solid excipient, optionally grinding a resulting mixture, andprocessing the mixture of granules, after adding suitable additionalcompounds, if desired, to obtain tablets or pills.

Suitable solid excipients are carbohydrate or protein fillers whichinclude, but are not limited to, sugars, including lactose, sucrose,mannitol, or sorbitol; starch from corn, wheat, rice, potato, or otherplants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; andgums including arabic and tragacanth; as well as proteins such asgelatin and collagen. If desired, disintegrating or solubilizing agentsmay be added, such as the cross-linked polyvinyl pyrrolidone, agar,alginic acid, or a salt thereof, such as sodium alginate. Pharmaceuticalpreparations that can also be used orally are, for example, push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating such as glycerol or sorbitol. Push-fit capsulescan contain ligands mixed with a filler or binders such as lactose orstarches, lubricants such as talc or magnesium stearate, and,optionally, stabilizers. In soft capsules, the ligands may be dissolvedor suspended in suitable liquids, such as fatty oils, liquid paraffin,or liquid polyethylene glycol with or without stabilizers.

Aqueous suspensions for internal use contain ligands mixed withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylnethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethylene oxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol (e.g.,polyoxyethylene sorbitol mono-oleate), or a condensation product ofethylene oxide with a partial ester derived from fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). Theaqueous suspension can also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate, and other additives as desired,including coloring agents, flavoring agents and sweetening agents, suchas sucrose, aspartame or saccharin. Formulations can be adjusted forosmolarity.

Oil suspensions for internal use can be formulated by suspending ligandsin a vegetable oil, such as arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilsuspensions can contain a thickening agent, such as beeswax, hardparaffin or cetyl alcohol. Sweetening agents can be added to provide apalatable oral preparation. These formulations can be preserved by theaddition of an antioxidant such as ascorbic acid.

Dispersible powders and granules of the disclosure suitable forpreparation of an aqueous suspension by the addition of water can beformulated from ligands in admixture with a dispersing, suspendingand/or wetting agent, and one or more preservatives. Suitable dispersingor wetting agents and suspending agents include those disclosed above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present.

The pharmaceutical formulations can also be in the form of oil-in-wateremulsions. The oily phase can be a vegetable oil, such as olive oil orarachis oil, a mineral oil, such as liquid paraffin, or a mixture ofthese. Suitable emulsifying agents include naturally-occurring gums,such as gum acacia and gum tragacanth, naturally occurring phosphatides,such as soybean lecithin, esters or partial esters derived from fattyacids and hexitol anhydrides, such as sorbitan mono-oleate, andcondensation products of these partial esters with ethylene oxide, suchas polyoxyethylene sorbitan mono-oleate. The emulsion can also containsweetening and flavoring agents. Syrups and elixirs can be formulatedwith sweetening agents, such as glycerol, sorbitol or sucrose. Suchformulations can also contain a demulcent, a preservative, a flavoringor a coloring agent.

For intravenous injection, water soluble antibodies can be administeredby the drip method, whereby a pharmaceutical formulation containing theformulation and a physiologically acceptable excipients is infused.Physiologically acceptable excipients may include, for example, 5%dextrose, 0.9% saline, Ringer's solution or other suitable excipients.

In one embodiment, the formulation is administered via site-specific ortargeted local delivery techniques. Examples of site-specific ortargeted local delivery techniques include various implantable depotsources of the antibody or local delivery catheters, such as infusioncatheters, an indwelling catheter, or a needle catheter, syntheticgrafts, adventitial wraps, shunts, and stents or other implantabledevices, site specific carriers, direct injection, or directapplication. See, e.g., WO 00/53211 and U.S. Pat. No. 5,981,568.

In another embodiment of the present disclosure, an article ofmanufacture is provided which contains any of the pharmaceuticalcompositions and formulations described herein (e.g., comprising abinding agent) and provides instructions for its use and/orreconstitution. The article of manufacture comprises a container.Suitable containers include, for example, bottles, vials (e.g. dualchamber vials), syringes (such as dual chamber syringes) and test tubes.The container may be formed from a variety of materials such as glass orplastic. The container holds the formulation and the label on, orassociated with, the container may indicate directions forreconstitution and/or use. For example, the label may indicate that theformulation is reconstituted to particular protein concentrations. Thecontainer holding the formulation may be a multi-use vial, which allowsfor repeat administrations (e.g., from 2-6 administrations) of thereconstituted formulation. The article of manufacture may furthercomprise a second container comprising a suitable diluent (e.g. BWFI).Upon mixing of the diluent and the lyophilized formulation, the finalprotein concentration in the reconstituted formulation will generally beat least 50 mg/mL. The article of manufacture may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use.

2. Administration and Dosing Regimen of the Formulations

The formulations containing pharmaceutically active binding agent andother active ingredients can be administered in any conventionallyacceptable way including, but not limited to, by intravenous,intramuscular, intraperitoneal, intracerebrospinal, subcutaneous,intracutaneous, intraarticular, intrasynovial, intrathecal, intradermal,intratumoral, intranodal, intramedulla, oral, inhalation or topicalroutes; or it may be administered orally, by inhalation spray,topically, rectally, nasally, buccally, vaginally or via an implantedreservoir; and in any case, as a bolus or by continuous infusion over aperiod of time; or via injectable depot routes of administration such asusing 1-, 3-, or 6-month depot injectable or biodegradable materials andmethods Administration will vary with the pharmacokinetics and otherproperties of the drugs and the patient's condition.

Commercially available nebulizers for liquid formulations, including jetnebulizers and ultrasonic nebulizers are useful for administration.Liquid formulations can be directly nebulized and lyophilized powder canbe nebulized after reconstitution. Alternatively, binding agents can beaerosolized using a fluorocarbon formulation and a metered dose inhaler,or inhaled as a lyophilized and milled powder.

The subject to be treated by the methods described herein can be amammal, more preferably a human. Mammals include, but are not limitedto, farm animals, sport animals, pets, primates, horses, dogs, cats,mice, and rats.

The amount of binding agent alone or in combination with another agentthat is adequate to accomplish this is considered the therapeuticallyeffective dose. The dosing schedule and amounts, i.e., the “dosingregimen,” will depend upon a variety of factors, including the stage ofthe disease or condition, the severity of the disease or condition, theseverity of the adverse side effects, the general state of the patient'shealth, the patient's physical status, age and the like. In calculatingthe dosage regimen for a patient, the mode of administration is alsotaken into consideration. The dosing regimen must also take intoconsideration the pharmacokinetics, i.e., the rate of absorption,bioavailability, metabolism, clearance, and the like. Based on thesevalues (which are determined in vitro, and in mammalian animal modelsand extrapolated to humans) the dosing regimen is projected for humans,and is then tested and further refined in clinical trials, in aconventional dose-finding study, as is well-known in the art.

The state of the art allows the clinician to determine the dosingregimen for each individual patient, depending on factors includingadministration route, disease stage, patient size, and patient level ofSARS-CoV2 or related pathogen. For example, a physician may initiallyuse escalating dosages, starting at a particular level, and then titratethe dosage at increments for each individual being treated based ontheir individual responses. Depending on the subject, the administrationof the formulation is maintained for as specific period of time or foras long as needed to effectively treat the subject's symptoms or preventtheir occurrence in the first place.

Lyophilized Formulation

After preparation of a suitable binding agent or conjugate, it can beprepared in a formulation for administration to a subject. A lyophilizedformulation is especially preferred for the binding agent, which as afirst step, requires preparing a pre-lyophilized formulation. The amountof binding agent in the pre-lyophilized formulation is determined takinginto account the desired dose volumes, mode(s) of administration etc.The binding agent is generally present in solution. For example, thebinding agent may be present in a pH-buffered solution at a pH fromabout 4-8, and preferably from about 5-7. Exemplary buffers includehistidine, phosphate, Tris, citrate, succinate and other organic acids.The buffer concentration can be from about 1 mM to about 20 mM, or fromabout 3 mM to about 15 mM, depending, for example, on the buffer and thedesired isotonicity of the formulation (e.g. of the reconstitutedformulation). The preferred buffer is histidine as it can havelyoprotective properties. Succinate is also a useful buffer.

The lyoprotectant is added to the pre-lyophilized formulation. Inpreferred embodiments, the lyoprotectant is a non-reducing sugar such assucrose or trehalose. The amount of lyoprotectant in the pre-lyophilizedformulation is generally such that, upon reconstitution, the resultingformulation will be isotonic, as preferred, though hypertonicreconstituted formulations may also be suitable. In addition, the amountof lyoprotectant must not be too low such that an unacceptable amount ofdegradation/aggregation of the Binding Agent occurs upon lyophilization.

Where the lyoprotectant is a sugar (such as sucrose or trehalose) andthe binding agent is an antibody, exemplary lyoprotectant concentrationsin the pre-lyophilized formulation are from about 10 mM to about 400 mM,and preferably from about 30 mM to 5 about 300 mM, and most preferablyfrom about 50 mM to about 100 mM.

The ratio of binding agent to lyoprotectant is selected for each bindingagent and lyoprotectant combination. In the case of an antibody as thebinding agent and a sugar (e.g., sucrose or trehalose) as thelyoprotectant for generating an isotonic reconstituted formulation witha high protein concentration, the molar ratio of lyoprotectant toantibody may be from about 100 to about 1500 moles lyoprotectant to 1mole antibody, and preferably from about 200 to about 1000 moles oflyoprotectant to 1 mole antibody, including from about 200 to about 600moles of lyoprotectant to 1 mole antibody.

In preferred embodiments, it has been found to be desirable to add asurfactant to the pre-lyophilized formulation. Alternatively, or inaddition, the surfactant may be added to the lyophilized formulationand/or the reconstituted formulation. Exemplary surfactants includenonionic surfactants such as polysorbates (e.g. polysorbates 20 or 80);poloxamers (e.g. poloxamer 188); Triton; sodium dodecyl sulfate (SDS);sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-,linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palnidopropyl-, or isostearamidopropyl-betaine (e.glauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g. Pluronics, PF68). The amount ofsurfactant added is such that it reduces aggregation of thereconstituted protein and minimizes the formation of particulates afterreconstitution. For example, the surfactant may be present in thepre-lyophilized formulation in an amount from about 0.001-0.5%, andpreferably from about 0.005-0.05%.

A mixture of the lyoprotectant (such as sucrose or trehalose) and abulking agent (e.g. mannitol or glycine) may be used in the preparationof the pre-lyophilization formulation. The bulking agent may allow forthe production of a uniform lyophilized cake without excessive pocketstherein.

Other pharmaceutically acceptable carriers, excipients or stabilizerssuch as those described in Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed. (1980) may be included in the pre-lyophilizedformulation (and/or the lyophilized formulation and/or the reconstitutedformulation) provided that they do not adversely affect the desiredcharacteristics of the formulation. Acceptable carriers, excipients orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed and include; additional buffering agents; preservatives;co-solvents; antioxidants including ascorbic acid and methionine;chelating agents such as EDTA; metal complexes (e.g. Zn-proteincomplexes); biodegradable polymers such as polyesters; and/orsalt-forming counterions such as sodium.

The pharmaceutical compositions and formulations described herein arepreferably stable, so as to retain its physical and chemical stabilityand integrity upon storage. Various analytical techniques for measuringprotein stability are available in the art and are reviewed in Peptideand Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker,Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev.10: 29-90 (1993). Stability can be measured at a selected temperaturefor a selected time period.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to, or following, lyophilization and reconstitution.Alternatively, sterility of the entire mixture may be accomplished byautoclaving the ingredients, except for protein, at about 120° C. forabout 30 minutes.

After the binding agent and lyoprotectant are mixed together, theformulation is lyophilized. Many different freeze-dryers are availablefor this purpose such as Hu1150® (Hull, USA) or GT20® (Leybold-Heraeus,Germany) freeze-dryers. Freeze-drying is accomplished by freezing theformulation and subsequently subliming ice from the frozen content at atemperature suitable for primary drying. Under this condition, theproduct temperature is below the eutectic point or the collapsetemperature of the formulation.

Typically, the shelf temperature for the primary drying will range fromabout −30 to 25° C. (provided the product remains frozen during primarydrying) at a suitable pressure, ranging typically from about 50 to 250mTorr. The formulation, size and type of the container holding thesample (e.g., glass vial) and the volume of liquid will mainly dictatethe time required for drying, which can range from a few hours toseveral days (e.g. 40-60 hrs). A secondary drying stage may be carriedout at about 0-40° C., depending primarily on the type and size ofcontainer and the type of protein employed. For example, the shelftemperature throughout the entire water removal phase of lyophilizationmay be from about 15-30° C. (e.g., about 20° C.). The time and pressurerequired for secondary drying will be that which produces a suitablelyophilized cake, dependent, e.g., on the temperature and otherparameters. The secondary drying time is dictated by the desiredresidual moisture level in the product and typically takes at leastabout 5 hours (e.g. 10-15 hours). The pressure may be the same as thatemployed during the primary drying step. Freeze-drying conditions can bevaried depending on the formulation and vial size.

In some instances, it may be desirable to lyophilize the proteinformulation in the container in which reconstitution of the protein isto be carried out in order to avoid a transfer step. The container inthis instance may, for example, be a 3, 5, 10, 20, 50 or 100 cc vial. Asa general proposition, lyophilization will result in a lyophilizedformulation in which the moisture content thereof is less than about 5%,and preferably less than about 3%.

At the desired stage, typically when it is time to administer theformulation to the patient, the lyophilized formulation may bereconstituted with a diluent such that the Binding Agent concentrationin the reconstituted formulation is preferably similar to that of thepre-lyophilized formulation.

Reconstitution generally takes place at a temperature of about 25° C. toensure complete hydration, although other temperatures may be employedas desired. The time required for reconstitution will depend, e.g., onthe type of diluent, amount of excipient(s) and protein. Exemplarydiluents include sterile water, bacteriostatic water for injection(BWFI), a pH buffered solution (e.g. phosphate-buffered saline), sterilesaline solution, Ringer's solution or dextrose solution. The diluentoptionally contains a preservative. Exemplary preservatives have beendescribed above, with aromatic alcohols such as benzyl or phenol alcoholbeing the preferred preservatives. The amount of preservative employedis determined by assessing different preservative concentrations forcompatibility with the protein and preservative efficacy testing. Forexample, if the preservative is an aromatic alcohol (such as benzylalcohol), it can be present in an amount from about 0.1-2.0% andpreferably from about 0.5-1.5%, but most preferably about 1.0-1.2%.

Alternatively, a non-lyophilized formulation may be used, including abinding agent, and any of the well-known carriers, excipients, buffers,stabilizers, preservatives, adjuvants and other additives describedherein and well known in the art.

Detection Assays

Provided methods permit detection of complex formation between a bindingagent and S-ECD-PFS. Detection of the complexes may be achieved by anyavailable method, e.g., an enzyme-linked immunosorbent assay (ELISA).For example, in some embodiments, an antibody to S-ECD-PFS is used. Insome embodiments, a secondary antibody, e.g., an anti-S-ECD-PFS antibodyis used. One or more antibodies may be coupled to a detection moiety. Insome embodiments, a detection moiety is or comprises a fluorophore. Asused herein, the term “fluorophore” (also referred to as “fluorescentlabel” or “fluorescent dye”) refers to moieties that absorb light energyat a defined excitation wavelength and emit light energy at a differentwavelength. In some embodiments, a detection moiety is or comprises anenzyme. In some embodiments, an enzyme is one (e.g., β-galactosidase)that produces a colored product from a colorless substrate.

As used herein, the terms “measuring” or “measurement,” or alternatively“detecting” or “detection,” means assessing the presence, absence,quantity or amount (which can be an effective amount) of a substancewithin a sample, including the derivation of qualitative or quantitativeconcentration levels of such substances, or otherwise evaluating thevalues or categorization of a subject's.

In some embodiments, a test is performed by adding capture agent to asubstrate, e.g., a reaction vessel, under conditions such that thecapture agent binds to the substrate, e.g., using an ELISA. A sample,e.g., tissue sample from a subject, blood, plasma, saliva or tears, maybe added to the capture-agent containing substrate in a reaction vessel.Any capture agent-binding molecules present may bind to the immobilizedcapture agent molecules. An antibody or an antibody-detection agentconjugate may be added to the reaction mixture. The antibody part of theconjugate binds to any antigen molecules, creating anantibody-antigen-antibody “sandwich.” After washing away any unboundconjugate, a substrate solution may be added to aid in detection. Forexample, after a set interval, the reaction may be stopped (e.g., byadding 1 N NaOH) and the concentration of colored product formed may bemeasured in a spectrophotometer. The intensity of color is proportionalto the concentration of bound antigen.

Examples and Experiments

It has been predicted the S protein contains a furin cleavage sitebetween R⁶⁸² and S⁶⁸⁶ (⁶⁸²RRAR↓S⁶⁸⁶), as shown in FIG. 1. The followingexperiments were carried out to verify furin's role in S proteincleavage.

S-ECD is Proteolytically Cleaved into Three Bands in Wild-Type HEK293Cells

To generate recombinant S-ECD proteins for uses in antibody development,first, a recombinant S-ECD having a 6-member Histamine tag protein atthe C terminus (“5-ECD-C6×His”) was produced in HEK293 cells, thenpurified using a nickel column. Three bands were observed for thewild-type S-ECD-C6×His following expression, purification and separationusing SDS-PAGE. As predicted, the results indicated that the S proteincontains a furin cleavage site between amino acid R682 and amino acidS686 (⁶⁸²RRAR↓S⁶⁸⁶). See FIG. 1. As shown in FIG. 2, the 180 KD bandcorresponds to S-ECD-PFS and the 120 KD and 80 KD bands seem tocorrespond to the mature/active form of the S protein containing the 51and S2 subunits.

However, the yield was low and the protein is not stable. The yield ofS-ECD in HEK293 wild-type cells is generally much lower (less than 1mg/1). Most of the recombinant S-ECDs on the market (referred to asS-ECD-MT) has a mutated cleavage site region, as shown in FIG. 1(⁶⁸²RRAR↓S⁶⁸⁶), in order to generate a product mimicking S-ECD-PFS, andto thereby avoid protease cleavage. However, the artificialmutation/deletion also generated an irrelevant epitope and ispotentially less useful in research or therapy. In addition, because ofthe mutation, the conventional recombinant protein is not suitable forfurther processing to generate an active form of furin.

Generation of Furin Gene KO Cells in HEK293 Cells by CRISPR

On the hypothesis that knocking out the FURIN gene in HEK293 cells andthen expressing S-ECD in the KO in HEK293 cells would generate an intactfurin protein, CRISPR-Cas9 was employed to generate the KO HEK293 cells.Two sgRNAs were designed to target exon 1. Single clones were selectedand the genomic DNA was sequenced and analyzed. Two clones weresequenced which both had a 56 bp deletion resulting in a frame-shift.The KO and WT sequences of furin in the relevant regions are depicted inFIG. 3 a.

Furin protein was assayed by Western Blot; and as expected, was nolonger present in the media from the KO HEK293 cells, as shown in FIG. 3b.

Increased Yield of S-ECD in HEK293 FURIN Gene KO Cells

The same construct of the S-ECD gene generating S-ECD-C6×His wastransfected into HEK293 wild type and Furin−/− cells. The conditionedmedia containing S-ECD-C6×His was separated by SDS-PAGE and detected byWestern Blot, using an anti-6×His antibody. As expected, recombinantS-ECD showed two bands in HEK293 wild type cells, one being thefull-length S-ECD and the other being S2, whereas recombinant S-ECDshowed a single band in furin−/− cells. Result are shown in FIG. 3 c.

To scale up the culture and transfection in furin−/− cells, conditionedmedia was collected to 1 L and proteins were purified with a nickelcolumn. The yield of S-ECD-PFS was much higher in furin−/− cellscompared with wild type HEK293 cells (>20 mg/l culture). Results areshown in FIG. 4.

S-ECD-PFS has Higher ACE2 Binding Affinity than S-ECD-MT

An enzyme-linked immunosorbent assay (ELISA) was used to measure andquantify the binding activity between the S-ECDs or recombinant S1 andACE2.

Recombinant S-ECDs and recombinant S1 (as a control) were immobilized at2 μg/mL (100 μL/well) to a 96 well plate. Recombinant ACE2 with an addedFc domain was added to the wells by 5-fold serial dilution, from 2 μg/mL(100/well), 6 times, and a blank was used as a negative control.Captured recombinant ACE2-Fc was measured by anti-human Fc-HRP antibody.The OD was measured and the EC50 was calculated by Origin8 software. TheEC50 of S1 was 12.14 ng/mL. The EC50 of S-ECD-MT was 10.38 ng/mL. TheEC50 of S-ECD-PFS was 4.33 ng/mL. Lower EC50 indicates the increasedbinding activity, as shown in FIG. 5.

These results indicate S-ECD-PFS has higher affinity and is superior toS1 or S-ECD-MT for use as an antigen to generate antibodies or for usein detection assays or in diagnosis, as a therapeutic to interfere withSARS-CoV-2 cellular binding, or as a vaccine (to generate antibodies toSARS-CoV-2).

Detailed Methods

The CRISPR Cas9 protocol used the following sgRNAs:

sgRNA1: (SEQ ID NO: 19) GATGCGCACAGCCCACGTGT sgRNA2: (SEQ ID NO: 20)ACAGTGTGGCACGGAAGCATincorporated into in the vector: £° PX459£¬addgene #62988 (from Addgene,Watertown Mass.) at the Bbs1 site. The forward and reverse sgRNAconstructs (with vectors) are shown below:

Vector-sgRNA1 (forward): (SEQ ID NO: 5) £° CACC-GATGCGCACAGCCCACGTGTVector-sgRNA1 (reverse): (SEQ ID NO: 6) £° AAAC-ACACGTGGGCTGTGCGCATCVector-sgRNA2 (forward): (SEQ ID NO: 7) £° CACC-ACAGTGTGGCACGGAAGCATVector-sgRNA2 (reverse): (SEQ ID NO: 8) £° AAAC-ATGCTTCCGTGCCACACTGT

For mammalian cell transfection and protein purification, plasmidpreparation was in accordance with the following exemplary protocol.

Plasmids all carry a betalactamase (amp) resistance gene and are grownin E. coli at 37° C. (or 30° C.) in shaker flasks overnight. Highquality plasmid DNA can be obtained using commercially availableMaxiprep kits (Qiagen), preferably including an endotoxin removal step.

HEK293 cells were adapted to Expi293 Expression Medium. The media andtransfection agents below were used in a standard protocol with theequipment below, but other cell lines (including 293T, CHO etc.) withother media and transfection reagents can also be used.

Materials and Equipment

-   -   Expi293 Expression Medium (Gibco #A1435102)    -   Opti-MEM I Reduced Serum Medium (Gibco #31985088)    -   ExpiFectamine 293 Transfection Kit (Glbco #A14524)    -   PBS (1×) (Gibco #10010-023 or equivalent)    -   Ni-NTA Agarose (Qiagen #30230 or equivalent)    -   NuPAGE 4-12% Bis-Tris Protein Gels (Invitrogen Catalog number:        NP0329BOX)    -   SDS-PAGE cell and power supply    -   Sodium Chloride NaCl (Sigma-Aldrich #S3014 or equivalent)    -   Imidazole (Sigma-Aldrich #I5513 or equivalent)    -   Furin mAb antibody (Abclonal A5043)    -   Actin mAb antibody (Abclonal ACO26)    -   Recombinant S-ECD R683A, R685A mutation (Abclonal RP01260MT)    -   Recombinant ACE2 (Abclonal RP01275)

Purification was performed with a nickel column (though other proteinpurification methods can also be used).

SequencesWild type FURIN gene locus: part of NCBI Reference Sequence: NC_000015.10cctgcccgtctcggccccatgcccccaccagtcagccccgggccacaggcagtgagcaggcacctgggagccgaggccctgtgaccaggccaaggagacgggcgctccagggtcccagccacctgtcccccccatggagctgaggccctggagctatgggtggtagcagcaacaggaaccaggtcctgctagcagctgatgctcagggccagaaggtcttcaccaacacgtgggctgtgcgcatccctggaggcccagcggtggccaacagtgtggcacggaagcatgggacctcaacctgggccaggtaggtgacccccacaggacactgccagggggtgggaccagagaagacagggattctgggagcaggagctgaggccttgatgctcaggggcatctgggtagccggcatgactgggtggccatgagcaaagcacaggtggttcaggcaagcagca (SEQID NO: 9) FURIN gene genomic sequencing primersseq-F: TCCTCTCAGGGTCGGCACTC (SEQ ID NO: 10), seq-R:GCTGCTTGCCTGAACCACCT (SEQ ID NO: 11) FURIN gene locus after KOcgtctcggccccatgcccccaccagtcagccccgggccacaggcagtgagcaggcacctgggagccgaggccctgtgaccaggccaaggagacgggcgctccagggtcccagccacctgtcccccccatggagctgaggccctggagctatgggtggtagcagcaacaggaaccaggtcctgctagcagctgatgctcagggccagaaggtatcaccaacacatgggacctcaacctgggccaggtaggtgacccccacaggacactgccagggggtgggaccagagaagacagggattctgggagcaggagctgttggccttgtttgctcaggggcatctgggtagccggcatgttctgggtggccatgagcaaagcacaggtggttcaggcaagcagca (SEQ ID NO: 12) Furin-WT-protein sequenceMELRPWLLWVVAATGTLVLLAADAQGQKVFTNTWAVRIPGGPAVANSVARKHGFLNLGQIFGDYYHFWHRGVTKRSLSPHRPRHSRLQREPQVQWLEQQVAKRRTKRDVYQEPTDPKFPQQWYLSGVTQRDLNVKAAWAQGYTGHGIVVSILDDGIEKNHPDLAGNYDPGASFDVNDQDPDPQPRYTQMNDNRHGTRCAGEVAAVANNGVCGVGVAYNARIGGVRMLDGEVTDAVEARSLGLNPNHIHIYSASWGPEDDGKTVDGPARLAEEAFFRGVSQGRGGLGSIFVWASGNGGREHDSCNCDGYTNSIYTLSISSATQFGNVPWYSEACSSTLATTYSSGNQNEKQIVTTDLRQKCTESHTGTSASAPLAAGIIALTLEANKNLTWRDMQHLVVQTSKPAHLNANDWATNGVGRKVSHSYGYGLLDAGAMVALAQNWTTVAPQRKCIIDILTEPKDIGKRLEVRKTVTACLGEPNHITRLEHAQARLTLSYNRRGDLAIHLVSPMGTRSTLLAARPHDYSADGFNDWAFMTTHSWDEDPSGEWVLEIENTSEANNYGTLTKFTLVLYGTAPEGLPVPPESSGCKTLTSSQACVVCEEGFSLHQKSCVQHCPPGFAPQVLDTHYSTENDVETIRASVCAPCHASCATCQGPALTDCLSCPSHASLDPVEQTCSRQSQSSRESPPQQQPPRLPPEVEAGQRLRAGLLPSHLPEVVAGLSCAFIVLVFVTVFLVLQLRSGFSFRGVKVYTMDRGLISYKGLPPEAWQEECPSDSEEDEGRGERTAFIKDQSAL (SEQ ID NO: 13) Furin-Mutation-protein sequenceMELRPWLLWVVAATGTLVLLAADAQGQKVFTNTW (SEQ ID NO: 14)* frame shiftS-ECD coding sequenceS-ECD (AA Val16-Gln1208 (SEQ ID NO: 15)) subcloned into pcDNA vector by5′XbaI/3′AgeI, after optimization (Accession #YP_009724390.1)gtgaacctgaccaccaggacccaacttcctcctgcctacaccaactccttcaccaggggagtctactaccctgacaaggtgttcaggtcctctgtgctgcacagcacccaggacctgttcctgccattcttcagcaatgtgacctggttccatgccatccatgtgtctggcaccaatggcaccaagaggtttgacaaccctgtgctgccattcaatgatggagtctactttgccagcacagagaagagcaacatcatcaggggctggatttttggcaccaccctggacagcaagacccagtccctgctgattgtgaacaatgccaccaatgtggtgattaaggtgtgtgagttccagttctgtaatgacccattcctgggagtctactaccacaagaacaacaagtcctggatggagtctgagttcagggtctactcctctgccaacaactgtacctttgaatatgtgagccaaccattcctgatggacttggagggcaagcagggcaacttcaagaacctgagggagtttgtgttcaagaacattgatggctacttcaagatttacagcaaacacacaccaatcaacctggtgagggacctgccacagggcttctctgccttggaaccactggtggacctgccaattggcatcaacatcaccaggttccagaccctgctggctctgcacaggtcctacctgacacctggagactcctcctctggctggacagcaggagcagcagcctactatgtgggctacctccaaccaaggaccttcctgctgaaatacaatgagaatggcaccatcacagatgctgtggactgtgccctggacccactgtctgagaccaagtgtaccctgaaatccttcacagtggagaagggcatctaccagaccagcaacttcagggtccaaccaacagagagcattgtgaggtttccaaacatcaccaacctgtgtccatttggagaggtgttcaatgccaccaggtttgcctctgtctatgcctggaacaggaagaggattagcaactgtgtggctgactactctgtgctctacaactctgcctccttcagcaccttcaagtgttatggagtgagcccaaccaaactgaatgacctgtgtttcaccaatgtctatgctgactcctttgtgattaggggagatgaggtgagacagattgcccctggacaaacaggcaagattgctgactacaactacaaactgcctgatgacttcacaggctgtgtgattgcctggaacagcaacaacctggacagcaaggtgggaggcaactacaactacctctacagactgacaggaagagcaacctgaaaccatttgagagggacatcagcacagagatttaccaggctggcagcacaccatgtaatggagtggagggcttcaactgttactaccactccaatcctatggatccaaccaaccaatggagtgggctaccaaccatacagggtggtggtgctgtcattgaactgctccatgcccctgccacagtgtgtggaccaaagaagagcaccaacctggtgaagaacaagtgtgtgaacttcaacttcaatggactgacaggcacaggagtgctgacagagagcaacaagaagacctgccattccaacagtaggcagggacattgctgacaccacagatgctgtgagggacccacagaccaggagattctggacatcacaccatgacctaggaggagtgtctgtgattacacctggcaccaacaccagcaaccaggtggctgtgctctaccaggatgtgaactgtactgaggtgcctgtggctatccatgctgaccaacttacaccaacctggagggtctacagcacaggcagcaatgtgttccagaccagggctggctgtctgattggagcagagcatgtgaacaactcctatgagtgtgacatcccaattggagcaggcatctgtgcctcctaccagacccagaccaacagcccaaggagggcaaggtctgtggcaagccagagcatcattgcctacacaatgagtctgggagcagagaactctgtggcttacagcaacaacagcattgccatcccaaccaacttcaccatctctgtgaccacagagattctgcctgtgagtatgaccaagacctctgtggactgtacaatgtatatctgtggagacagcacagagtgtagcaacctgctgctccaatatggctccactgtacccaacttaacagggctctgacaggcattgctgtggaacaggacaagaacacccaggaggtgatgcccaggtgaagcagatttacaagacacctccaatcaaggactaggaggcttcaacttcagccagattctgcctgacccaagcaagccaagcaagaggtcatcattgaggacctgctgacaacaaggtgaccctggctgatgctggcttcatcaagcaatatggagactgtctgggagacattgctgccagggacctgatagtgcccagaagttcaatggactgacagtgctgcctccactgctgacagatgagatgattgcccaatacacctctgccctgctggctggcaccatcacctctggctggacctaggagcaggagcagccctccaaatcccatttgctatgcagatggcttacaggacaatggcattggagtgacccagaatgtgctctatgagaaccagaaactgattgccaaccagttcaactctgccattggcaagattcaggactccctgtccagcacagcctctgccctgggcaaactccaagatgtggtgaaccagaatgcccaggctctgaacaccctggtgaagcaactaccagcaactaggagccatctcctctgtgctgaatgacatcctgagcagactggacaaggtggaggctgaggtccagattgacagactgattacaggcagactccaatccctccaaacctatgtgacccaacaacttatcagggctgctgagattagggcatctgccaacctggctgccaccaagatgagtgagtgtgtgctgggacaaagcaagagggtggacactgtggcaagggctaccacctgatgagattccacagtctgcccctcatggagtggtgacctgcatgtgacctatgtgcctgcccaggagaagaacttcaccacagcccctgccatctgccatgatggcaaggctcactaccaagggagggagtgatgtgagcaatggcacccactggtagtgacccagaggaacactatgaaccacagattatcaccacagacaacacctagtgtctggcaactgtgatgtggtgattggcattgtgaacaacacagtctatgacccactccaacctgaactggactcatcaaggaggaactggacaaatacttcaagaaccacaccagccctgatgtggacctgggagacatctctggcatcaatgcctctgtggtgaacatccagaaggagattgacagactgaatgaggtggctaagaacctgaatgagtccctgattgacctccaagaactgggcaaatatgaacaatacatcaagtggccacatcatcaccaccatcactaaS-ECD (AA Val16-Gln1208 (SEQ ID NO: 16)) Protein sequenceVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQHHHHHH 6. S-ECD cloning sequenceS-ECD (AA Met1-Gln1208 (SEQ ID NO: 17)) which was subcloned into pcDNAvector by 5′XbaI/3′AgeI; includes the optimized DNA codon; with the underlinedsignal peptide coding section (Accession #YP_009724390.1)atgtttgtgttcctggtgctgctgccactggtgtccagccagtgtgtgaacctgaccaccaggacccaacttcctcctgcctacaccaactccttcaccaggggagtctactaccctgacaaggtgttcaggtcctctgtgctgcacagcacccaggacctgttcctgccattcttcagcaatgtgacctggttccatgccatccatgtgtctggcaccaatggcaccaagaggtttgacaaccctgtgctgccattcaatgatggagtctactttgccagcacagagaagagcaacatcatcaggggctggatttttggcaccaccctggacagcaagacccagtccctgctgattgtgaacaatgccaccaatgtggtgattaaggtgtgtgagttccagttctgtaatgacccattcctgggagtctactaccacaagaacaacaagtcctggatggagtctgagttcagggtctactcctctgccaacaactgtacctttgaatatgtgagccaaccattcctgatggacttggagggcaagcagggcaacttcaagaacctgagggagtttgtgttcaagaacattgatggctacttcaagatttacagcaaacacacaccaatcaacctggtgagggacctgccacagggcttctctgccttggaaccactggtggacctgccaattggcatcaacatcaccaggttccagaccctgctggctctgcacaggtcctacctgacacctggagactcctcctctggctggacagcaggagcagcagcctactatgtgggctacctccaaccaaggacchcctgctgaaatacaatgagaatggcaccatcacagatgctgtggactgtgccctggacccactgtctgagaccaagtgtaccctgaaatccttcacagtggagaagggcatctaccagaccagcaacttcagggtccaaccaacagagagcattgtgaggtttccaaacatcaccaacctgtgtccatttggagaggtgttcaatgccaccaggtttgcctctgtctatgcctggaacaggaagaggattagcaactgtgtggctgactactctgtgctctacaactctgcctccttcagcaccttcaagtgttatggagtgagcccaaccaaactgaatgacctgtgtttcaccaatgtctatgctgactcctttgtgattaggggagatgaggtgagacagattgcccctggacaaacaggcaagattgctgactacaactacaaactgcctgatgacttcacaggctgtgtgattgcctggaacagcaacaacctggacagcaaggtgggaggcaactacaactacctctacagactgttcaggaagagcaacctgaaaccatttgagagggacatcagcacagagatttaccaggctggcagcacaccatgtaatggagtggagggcttcaactgttactttccactccaatcctatggcttccaaccaaccaatggagtgggctaccaaccatacagggtggtggtgctgtcctttgaactgctccatgcccctgccacagtgtgtggaccaaagaagagcaccaacctggtgaagaacaagtgtgtgaacttcaacttcaatggactgacaggcacaggagtgctgacagagagcaacaagaagttcctgccattccaacagtttggcagggacattgctgacaccacagatgctgtgagggacccacagaccttggagattctggacatcacaccatgttcctttggaggagtgtctgtgattacacctggcaccaacaccagcaaccaggtggctgtgctctaccaggatgtgaactgtactgaggtgcctgtggctatccatgctgaccaacttacaccaacctggagggtctacagcacaggcagcaatgtgttccagaccagggctggctgtctgattggagcagagcatgtgaacaactcctatgagtgtgacatcccaattggagcaggcatctgtgcctcctaccagacccagaccaacagcccaaggagggcaaggtctgtggcaagccagagcatcattgcctacacaatgagtctgggagcagagaactctgtggcttacagcaacaacagcattgccatcccaaccaacttcaccatctctgtgaccacagagattctgcctgtgagtatgaccaagacctctgtggactgtacaatgtatatctgtggagacagcacagagtgtagcaacctgctgctccaatatggctccttctgtacccaacttaacagggctctgacaggcattgctgtggaacaggacaagaacacccaggaggtgtttgcccaggtgaagcagatttacaagacacctccaatcaaggactttggaggcttcaacttcagccagattctgcctgacccaagcaagccaagcaagaggtccttcattgaggacctgctgttcaacaaggtgaccctggctgatgctggcttcatcaagcaatatggagactgtctgggagacattgctgccagggacctgatttgtgcccagaagttcaatggactgacagtgctgcctccactgctgacagatgagatgattgcccaatacacctctgccctgctggctggcaccatcacctctggctggacctttggagcaggagcagccctccaaatcccatttgctatgcagatggcttacaggttcaatggcattggagtgacccagaatgtgctctatgagaaccagaaactgattgccaaccagttcaactctgccattggcaagattcaggactccctgtccagcacagcctctgccctgggcaaactccaagatgtggtgaaccagaatgcccaggctctgaacaccctggtgaagcaactttccagcaactttggagccatctcctctgtgctgaatgacatcctgagcagactggacaaggtggaggctgaggtccagattgacagactgattacaggcagactccaatccctccaaacctatgtgacccaacaacttatcagggctgctgagattagggcatctgccaacctggctgccaccaagatgagtgagtgtgtgctgggacaaagcaagagggtggacttctgtggcaagggctaccacctgatgagttttccacagtctgcccctcatggagtggtgttcctgcatgtgacctatgtgcctgcccaggagaagaacttcaccacagcccctgccatctgccatgatggcaaggctcactttccaagggagggagtgtttgtgagcaatggcacccactggtttgtgacccagaggaacttctatgaaccacagattatcaccacagacaacacctttgtgtctggcaactgtgatgtggtgattggcattgtgaacaacacagtctatgacccactccaacctgaactggactccttcaaggaggaactggacaaatacttcaagaaccacaccagccctgatgtggacctgggagacatctctggcatcaatgcctctgtggtgaacatccagaaggagattgacagactgaatgaggtggctaagaacctgaatgagtccctgattgacctccaagaactgggcaaatatgaacaatacatcaagtggccacatcatcaccaccatcactaaProtein sequence (AA Met1-Gln1208(SEQ ID NO: 18)), with signal peptide underlinedMFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQHHHHHH

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. Thus, for example, in eachinstance herein, in embodiments or examples of the present invention,any of the terms “comprising”, “including”, containing”, etc. are to beread expansively and without limitation. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and that they are not necessarily restricted to theorders of steps indicated herein or in the claims. It is also noted thatas used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference, and the plural include singularforms, unless the context clearly dictates otherwise. Under nocircumstances may the patent be interpreted to be limited to thespecific examples or embodiments or methods specifically disclosedherein. Under no circumstances may the patent be interpreted to belimited by any statement made by any Examiner or any other official oremployee of the Patent and Trademark Office unless such statement isspecifically and without qualification or reservation expressly adoptedin a responsive writing by Applicants.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. The terms and expressionsthat have been employed are used as terms of description and not oflimitation, and there is no intent in the use of such terms andexpressions to exclude any equivalent of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention as claimed.Thus, it will be understood that although the present invention has beenspecifically disclosed by preferred embodiments and optional features,modification and variation of the concepts herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention asdefined by the appended claims.

REFERENCES

-   1. Coutard B, Valle C, de Lamballerie X, Canard B, Seidah N G,    Decroly E. The spike glycoprotein of the new coronavirus 2019-nCoV    contains a furin-like cleavage site absent in CoV of the same clade.    Antiviral Res. 2020; 176:104742. doi:    10.1016/j.antivira1.2020.104742-   2. Hoffmann M, Kleine-Weber H, Schroeder S, et al. SARS-CoV-2 Cell    Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically    Proven Protease Inhibitor. Cell. 2020; 181(2):271-280.e8. doi:    10.1016/j.cell.2020.02.052-   3. Furin See Wikipedia Website-   4. Stadlbauer D, Amanat F, Chromikova V, et al. SARS-CoV-2    Seroconversion in Humans: A Detailed Protocol for a Serological    Assay, Antigen Production, and Test Setup. Curr Protoc Microbiol.    2020; 57(1):e100. doi:10.1002/cpmc.100

What is claimed is:
 1. A method of generating binding agents tocoronavirus spike proteins which were generated recombinantly in apre-fusion state, comprising: generating host cells which lack theability to express furin but which express SARS-CoV-2 spike protein;expressing the SARS-CoV-2 spike protein in said host cells; andgenerating binding molecules to the SARS-CoV-2 spike protein.
 2. Themethod of claim 1 wherein the coronaviruses are SARS-CoV-2, CoV-ZXC21,SARS-CoV, SARS-like BM4821, HCoV-OC43, HKU9-1, HCoV-NL63, HCoV229E,MERS-CoV and HKU1.
 3. The method of claim 1 wherein binding agents aremonoclonal antibodies generated in mice, rat, human, hamster, goat,llama, alpaca or rabbit cells.
 4. The method of claim 1 wherein thebinding agents are chimeric, humanized or human monoclonal antibodies;Fab, Fab′, F(ab)′2 or Fv fragments; single domain antibodies;helix-stabilized antibodies; diabodies; disulfide stabilized antibodies;single-chain antibody molecules; domain antibodies; or bi-specificantibodies.
 5. The method of claim 1 wherein the host cells are furin−/− knock out cells.
 6. The method of claim 1 wherein the host cellsHEK293 cells.
 7. The method of claim 1 wherein a CRISPR Cas9transfection protocol is used to generate genetically modified cellswhich lack the ability to express furin.
 8. The method of claim 7wherein the transfection protocol uses the following sgRNAs:(SEQ ID NO: 19) GATGCGCACAGCCCACGTGT; and (SEQ ID NO: 20)ACAGTGTGGCACGGAAGCAT.


9. The method of claim 8 wherein the sgRNAs are incorporated into in avector.
 10. A method of treating or preventing Covid-19 comprising:generating recombinant SARS-CoV-2 spike protein in a pre-fusion stateusing furin knock out cells as hosts; generating binding agents to therecombinant SARS-CoV-2 spike protein in pre-fusion state; andadministering an effective amount of said binding agents to prevent ortreat Covid-19.
 11. The method of claim 10 wherein the binding agentsare monoclonal antibodies generated in mice, rat, human, hamster, goat,llama, alpaca or rabbit cells.
 12. The method of claim 10 wherein thebinding agents are chimeric, humanized or human monoclonal antibodies;Fab, Fab′, F(ab)′2 or FIT fragments; single domain antibodies;helix-stabilized antibodies; diabodies; disulfide stabilized antibodies;single-chain antibody molecules; domain antibodies; or bi-specificantibodies.
 13. The method of claim 10 wherein the host cells HEK293cells.
 14. The method of claim 10 wherein a CRISPR Cas9 transfectionprotocol is used to generate genetically modified cells which lack theability to express furin.
 15. The method of claim 14 wherein thetransfection protocol uses the following sgRNAs: (SEQ ID NO: 19)GATGCGCACAGCCCACGTGT; and (SEQ ID NO: 20) ACAGTGTGGCACGGAAGCAT.


16. The method of claim 15 wherein the sgRNAs are incorporated into in avector.
 17. A method of detecting SARS-CoV-2 in blood products fortransfusion, or in tissues for transplantation, comprising: generatingrecombinant SARS-CoV-2 spike protein in a pre-fusion state using furinknock out cells as hosts; generating binding agents to the recombinantSARS-CoV-2 spike protein in pre-fusion state; and using the bindingagents to assay blood products or tissues for SARS-CoV-2.
 18. The methodof claim 17 wherein the binding agents are monoclonal antibodiesgenerated in mice, rat, human, hamster, goat, llama, alpaca or rabbitcells.
 19. The method of claim 17 wherein the binding agents arechimeric, humanized or human monoclonal antibodies; Fab, Fab′, F(ab)′2or Fv fragments; single domain antibodies; helix-stabilized antibodies;diabodies; disulfide stabilized antibodies; single-chain antibodymolecules; domain antibodies; or bi-specific antibodies.
 20. The methodof claim 17 wherein the host cells HEK293 cells.